Analysis of Protein Post-Translational Modifications by Mass Spectrometry

John R. Griffiths, Richard D. Unwin

• Publisher: Wiley
• Year: 2016
• ISBN:
  9781119250890
• Edition: 1
• Topic: Naturwissenschaften
• Subtopic: Spektroskopie & Spektralanalyse

• Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation
• Discussion of the chemistry behind each modification, along with key methods and references
• Contributions from some of the leading researchers in the field
• A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

Chapter 1
Introduction

Rebecca Pferdehirt, Florian Gnad and Jennie R. Lill

Proteomics and Biological Resources, Genentech Inc., South San Francisco, CA, USA

1.1 Post-translational Modification of Proteins

While the human proteome is encoded by approximately 20,000 genes [1, 2], the functional diversity of the proteome is orders of magnitude larger because of added complexities such as genomic recombination, alternative transcript splicing, or post-translational modifications (PTMs) [3, 4]. PTMs include the proteolytic processing of a protein or the covalent attachment of a chemical or proteinaceous moiety to a protein allowing greater structural and regulatory diversity. Importantly, PTMs allow for rapid modification of a protein in response to a stimulus, resulting in functional flexibility on a timescale that traditional transcription and translation responses could never accommodate. PTMs range from global modifications such as phosphorylation, methylation, ubiquitination, and glycosylation, which are found in all eukaryotic species in all organs, to more specific modifications such as crotonylation (thought to be spermatozoa specific) and hypusinylation (specific for EIF5a), which govern more tight regulation of associated proteins. Taken together, over 200 different types of PTMs have been described [5], resulting in an incredibly complex repertoire of modified proteins throughout the cell.

The addition and subtraction of PTMs are controlled by tight enzymatic regulation. For example, many proteins are covalently modified by the addition of a phosphate group onto tyrosine, serine, or threonine residues in a process called phosphorylation [6]. Phosphorylation is catalyzed by a diverse class of enzymes called kinases [7], whereas these phosphomoieties are removed by a second class of enzymes referred to as phosphatases. The tight regulation of kinases and phosphatases often creates “on/off” switches essential for regulation of sensitive signaling cascades. There are some exceptions to this rule however, and the hunt is still underway for the ever-elusive hypusine [8] removing enzyme or putative enzymes responsible for the removal of protein arginine methylation. However, it is also possible that proteins bearing these PTMs are modulated or removed from the cell by other mechanisms of action. For example, proteolysis is rarely (if ever) reversible, and many proteins (e.g., blood clotting factors and digestive enzymes) are tightly governed by irreversible cleavage events where the active form is created after proteolysis of a proenzyme.

While PTMs such as phosphorylation and lysine acetylation exist in a binary “on/off” state, many other PTMs exhibit much more complex possible modification patterns. For example, lysine residues can be modified by covalent attachment of the small protein ubiquitin, either by addition of a single ubiquitin or by addition of ubiquitin polymers. In the latter case ubiquitin itself is used as the point of attachment for addition of subsequent ubiquitin monomers [9]. To add another layer of complexity, ubiquitin has seven lysines (K6, K11, K27, K29, K33, K48, and K63), each of which may be used as the point of polyubiquitin chain linkage, and each of which has a different functional consequence. For example, K63-linked chains are associated with lysosomal targeting, whereas K48-linked chains trigger substrate degradation by the proteasome. Thus, even within one type of PTM, multiple subtypes exist, further expanding the functional possibilities of protein modification.

In addition, many proteins are modified on multiple residues by different types of PTMs. A classic example is the PTM of histones. Histones are nuclear proteins that package and compact eukaryotic DNA into structural units called nucleosomes, which are the basic building blocks of chromatin and essential for regulation of gene expression. The C-termini of histones are composed of unstructured tails that protrude from nucleosomes and are heavily modified by methylation, acetylation, ubiquitylation, phosphorylation, SUMOylation, and other PTMs [10]. Overall, 26 modified residues on a single-core histone have been identified, and many of these residues can harbor multiple PTM types. In a generally accepted theory referred to as the “histone code,” the combination of PTMs on all histones comprising a single nucleosome or group of nucleosomes regulates fine-tuned expression of nearby genes.

As we begin to uncover the modified proteome, the importance of the interplay between multiple different PTMs has become increasingly apparent. One classic example is the involvement of both protein phosphorylation and ubiquitylation in the regulation of signaling networks [11]. Protein phosphorylation commonly promotes subsequent ubiquitylation, and the activities of ubiquitin ligases are also frequently regulated through phosphorylation. In a recent study by Ordureau et al., quantitative proteomic studies were employed to describe the PINK1 kinase–PARKIN UB ligase pathway and its disruption in Parkinson’s disease [12]. The authors describe a feedforward mechanism where phosphorylation of PARKIN by PINK1 occurs upon mitochondrial damage, leading to ubiquitylation of mitochondria and mitochondrial proteins by PARKIN. These newly formed ubiquitin chains are then themselves phosphorylated by PINK1, which promotes association of phosphorylated PARKIN with polyubiquitin chains on the mitochondria, and ultimately results in signal amplification. This model exemplifies how intricate interactions between multiple different PTMs regulate protein localization, interactions, activity, and ultimately essential cellular processes.

Recent advances in mass spectrometry methods, instrumentation, and bioinformatics analyses have enabled the identification and quantification of proteome-wide PTMs. For example, it is now a common practice to identify ten thousand phosphorylation sites in a single phosphoproteome enrichment experiment [13]. In addition, precise quantitation allows a deeper understanding of the combinations and occupancy of PTMs within a given protein. Such MS-based PTM analyses have led to previously impossible discoveries, advancing our understanding of the role of PTMs in diverse biological processes.

1.2 Global versus Targeted Analysis Strategies

Detection of PTMs by mass spectrometry can be achieved via global or targeted methods. The biological pathway of interest usually determines the type of PTM to be analyzed and associated methods. In a more targeted approach, researchers decide to investigate PTMs, because a protein of interest shows a higher than expected molecular weight or multiple bands by western blot after application of a stimulus, thus prompting speculation as to whether this could be due to PTM. Either way, the first step in PTM mapping is to determine the type of PTM of interest. In some cases the observed mass shift in a mass spectrometer indicates a certain PTM type. Many PTMs, however, result in the same mass addition (e.g., +42 Da for both acetylation and trimethylation). One powerful strategy in determining PTM identity involves the employment of the enzymes responsible for PTM removal. For example, after antibody enrichment of a modified protein, the antibody-bound protein can be incubated with general phosphatases, deubiquitinating enzymes (DUBs), or deSUMOylating enzymes (SENPs), and PTM removal can be assayed by western blot. Another method for PTM identification is western blotting with PTM specific “pan-antibodies.” Many commercially available antibodies exist for this purpose, recognizing common PTMs such as acetylation, methylation, ubiquitylation, and phosphorylation or even more rare PTMs such as crotonyl-, malonyl- or glutaryl-lysine modification. Once the type of PTM that is decorating a protein has been identified, the next step is to attempt to map the amino acid residue(s) that bear this modification.

One of the first applications of mass spectrometry in protein research was the mapping of a PTM on a single protein [14]. A commonly used approach involves protein-level immunoprecipitation followed by separating the captured proteins by SDS-PAGE, excising the higher molecular weight band, and performing in-gel tryptic digestion followed by LC-MS/MS. By searching for mass shifts indicative of the suspected modification(s), PTM-containing peptides can be identified and the PTM site mapped back to the protein. The strategy of identifying proteins in complex mixtures by digesting them into peptides, sequencing the resulting peptides by tandem mass spectrometry (MS/MS), and determining peptide and protein identity through automated database searching is referred to as shotgun proteomics and is one of the most popular analysis strategies in proteomics [15]. This protein-level enrichment approach, however, is dependent on sufficient levels of the modified protein compared to unmodified and the availability of protein-specific antibodies for immunoprecipitation. It is also possible that modifications may occur within the antibody epitope, blocking enrichment of the modified form altogether.

Researchers are commonly interested in analyzing PTMs from a complex mixture of proteins rather than on only one substrate. This can be a challenge, since modified peptides often occur in substoichiometric levels compared to unmodified versions and also may ionize less efficiently by electrospray ionization (ESI). However, several enrichment strategies exist, allowing for reduction of sample complexity and easier detection of the modified peptide species. Peptide-level immunoprecipitation using antibodies specific to a given PTM is an increasingly popular method of enrichment prior to MS. While this strategy can be employed for any PTM enrichment, it has been most commonly used for mapping ubiquitination sites. Tryptic digestion of ubiquitinated proteins generates a diglycine remnant attached to the ubiquitinated lysine residue (K-GG) that can be recognized by antibodies. The resulting mass shift of +114.0429 Da can be detected by MS/MS. Not only has K-GG peptide immunoaffinity enrichment enabled the identification of hundreds of ubiquitination sites o...